In recent years, efforts have increasingly been made to produce industrially complex carbohydrates, such as secreted oligosaccharides. This has been due to the roles of such compounds in numerous biological processes in living organisms. Secreted oligosaccharides, such as human milk oligosaccharides (HMOs), have become particularly important commercial targets for nutrition and therapeutic applications. Human milk oligosaccharides have become of great interest in the past few years due to their important functions in human development. To date, the structures of more than 140 HMOs have been determined, and considerably more are probably present in human milk (Urashima et al.: Milk oligosaccharides, Nova Science, 2011; Chen Adv. Carbohydr. Chem. Biochem. 72, 113 (2015)).
To date, ways of making large volumes of human milk oligosaccharides at low cost have not been available. The isolation of oligosaccharides from human milk has been rather difficult, even in milligram quantities, and very expensive due to the presence of a large number of other similar oligosaccharides in human milk. This problem has not been solved by current biotechnology or synthetic chemistry technology with regard to higher HMOs. Because of the growing commercial interest in nutritional compositions and supplements containing HMOs, there has been a need for a low cost method of making such HMOs.
Para-lacto-N-neohexaose (pLNnH) was proposed to be a core HMO, the sialylated and/or fucosylated derivatives of which are mother milk components ((Urashima et al.: Milk oligosaccharides, Nova Science, 2011). Recently, pLNnH itself has been shown to be present in human milk (Yu et al. Mol. Cell. Proteom. 13, 2944 (2014), Ashline et al. Mol. Cell. Proteom. 13, 2961 (2014)).
The chemical synthesis of pLNnH has been disclosed by Shimizu et al. (Bioorg. Med. Chem. Lett. 6, 2841 (1996)), by Ali et al. (Eur. J. Org. Chem. 319 (2000)) and in WO 2013/044928. The in vitro enzymatic synthesis of pLNnH has been described by Di Virgilio et al. (Glycobiology 9, 353 (1999)). The one-cell biosynthesis of pLNnH, together with LNnT and other higher oligosaccharides, has been reported in WO 01/04341, by Priem et al. (Glycobiology 12, 235 (2002)) and by Gebus et al. (Carbohydr. Res. 361, 83 (2012)).
Crystalline HMOs, produced in industrial amounts, are highly desirable for food and medical applications. However, no crystalline form of pLNnH has, as yet, been described. In addition, the above prior art methods are not suitable for scaling-up and industrial production of pLNnH.
Crystallization or recrystallization is one of the simplest and cheapest methods to separate a chemical product from contaminants and obtain it in pure form. In addition, crystalline modifications of a solid compound are important in the development of a compound, because different crystalline forms or polymorphs can have different properties—for example its thermodynamic stability, solubility, density and hygroscopicity.